TeaTime4schools: joint analysis - fungi

set.seed(1000)
min_lib_size <- 5000
metadata_path <- "./"
data_path <- "./DADA2_pseudo/"
Metadata_table <- "TeaTime_joint_Fungi_metadata.csv"
Seq_table <- "DADA2.seqtab_nochim.tsv"
Tax_table <- "DADA2.taxa_unite.tsv"
Proj_name <- "TeaTime4Schools"

# read OTU mat from data file
Raw_data <- read_tsv(paste0(data_path, Seq_table), 
                        trim_ws = TRUE)
contaminant_seqs <- read_csv(paste0(data_path, "decontam_contaminants.csv"), 
                        trim_ws = TRUE,
                        col_names = FALSE)

Raw_data %<>% # remove contaminant OTUs. 
  # .[, -grep("CTRL", colnames(.))] %>% # remove ext. cont. 
  .[!(Raw_data$`#OTU` %in% contaminant_seqs$X1), ] 

Raw_data[, 2:ncol(Raw_data)] %>% 
  t() %>% 
  as.data.frame() -> abundance_mat # convert to abundance matrix
colnames(abundance_mat) <- pull(Raw_data, "#OTU") # add sequence names

# Read metadata file
read_csv(paste0(metadata_path, Metadata_table),
         trim_ws = TRUE) %>%
  mutate_at(
    c(
      "Workshop",
      "Season",
      "Run",
      "Type",
      "Sample type",
      "Field",
      "Replicate",
      "Control",
      "Gene"
    ),
    ~factor(.)
  ) %>% 
  mutate_at(c("Extr. Date", "PCR products_ITS_send for seq"), ~as.Date(., "%d.%m.%Y")) ->
  Metadata
Metadata$Season %<>% fct_relevel("Winter", "Spring", "Summer", "Autumn")
Metadata$Read1_file <- str_replace(Metadata$Read1_file, "(.*)_L001_R1_001.fastq.gz|\\.1\\.fastq.gz", "\\1")
Metadata <- Metadata[Metadata$Read1_file %in% rownames(abundance_mat), ] # remove metadata rows if the samples did not go through qual processing

# Order abundance_mat samples according to the metadata
sample_order <- match(rownames(abundance_mat), Metadata$Read1_file)
# Order abundance_mat samples according to the metadata
sample_order <- match(rownames(abundance_mat), Metadata$Read1_file)
abundance_mat %<>% 
  rownames_to_column('sample_name') %>% 
  arrange(., sample_order) %>% 
  column_to_rownames('sample_name') # needed for phyloseq

Metadata$Library.size <- rowSums(abundance_mat)
Metadata <- data.frame(row.names = Metadata$Read1_file, Metadata)

# read taxonomy from data file
Raw_tax_data <- read_tsv(paste0(data_path, Tax_table), 
                        trim_ws = TRUE, col_names = TRUE)
Raw_tax_data %<>%
  .[!(Raw_tax_data$`Seq#` %in% contaminant_seqs$X1),] %>% # remove contaminant OTUs.
  mutate_all(funs(replace(., is.na(.), "Unclassified"))) # I think mutaute_all is unnecessary here because replace(., is.na(.), "Unclassified") alone should work

Raw_tax_data %>%
  select(.,
         Kingdom = V8,
         Phylum = V9,
         Class = V10,
         Order = V11,
         Family = V12,
         Genus = V13) %>%
  cbind(Name = colnames(abundance_mat),. ) ->
  Taxonomy.bs

Raw_tax_data %>%
  select(.,
         Kingdom,
         Phylum,
         Class,
         Order,
         Family,
         Genus)  %>% 
  map_dfr(~str_remove_all(., "^.__")) %>% # remove "k__" prefixes 
  add_column(Seq = colnames(abundance_mat)) %>% 
  column_to_rownames("Seq") %>% 
  set_colnames(c("Domain", "Phylum", "Class", "Order", "Family", "Genus")) %>% 
  as.matrix() -> # because tax_table() needs a matrix for retaining row.names
  Taxonomy

# generate phyloseq object
Ps_obj <- phyloseq(otu_table(abundance_mat, taxa_are_rows = FALSE),
                        tax_table(Taxonomy),
                        sample_data(Metadata)
                        )
Ps_obj <- filter_taxa(Ps_obj, function(x) sum(x) > 0, TRUE) # remove 0 abundance taxa
Ps_obj <- subset_samples(Ps_obj, sample_sums(Ps_obj) > 0) # remove 0 abundance samples
# Remove mock and control samples
Ps_obj <- subset_samples(Ps_obj, Type != "Control" & Type != "Mock")

# Create a grouping variable for merging
sample_data(Ps_obj) %<>% 
  as(., "data.frame") %>% 
  # get_variable(., c("Sample.type", "Field", "Season", "Replicate")) %>% 
  unite(., "Description", c("Sample.type", "Field", "Season", "Replicate"), remove = FALSE)
sample_data(Ps_obj)$Description %<>% as_factor(.)

# merged_Ps_obj <- merge_samples(Ps_obj, "Description")
# merged_SD <- merge_samples(sample_data(Ps_obj), "Description")
Ps_obj_merged <- MergeSamples(Ps_obj, grouping_name = "Description")

table(tax_table(Ps_obj_merged)[, "Domain"], exclude = NULL)

table(tax_table(Ps_obj_merged)[, "Class"], exclude = NULL)

# table(tax_table(Ps_obj_merged)[, "Family"], exclude = NULL)

domains2remove <- c("", "Bacteria", "Archaea", "Unclassified")
orders2remove <- c("")
families2remove <- c("")

Ps_obj_filt <- subset_taxa(Ps_obj_merged, !is.na(Phylum) &
                        !Domain %in% domains2remove &
                      !Order %in% orders2remove &
                      !Family %in% families2remove)


Taxonomy.bs %<>% 
  filter(Taxonomy.bs$Name %in% row.names(Ps_obj_filt@tax_table))
sample_data(Ps_obj_filt)$Lib.size <- rowSums(otu_table(Ps_obj_filt))

prevdf <- apply(X = otu_table(Ps_obj_filt),
                 MARGIN = ifelse(taxa_are_rows(Ps_obj_filt), yes = 1, no = 2),
                 FUN = function(x){sum(x > 0)})
# Add taxonomy and total read counts to this data.frame
prevdf <- data.frame(Prevalence = prevdf,
                      TotalAbundance = taxa_sums(Ps_obj_filt),
                      tax_table(Ps_obj_filt))

prevdf %>%
  group_by(Phylum) %>%
  summarise(`Mean prevalence` = mean(Prevalence),
            `Sum prevalence` = sum(Prevalence)) ->
  Prevalence_phylum_summary

Prevalence_phylum_summary %>% 
  kable(., digits = c(0, 1, 0)) %>%
  kable_styling(bootstrap_options = c("striped", "hover", "condensed", "responsive"), full_width = F)

prevdf %>%
  group_by(Order) %>%
  summarise(`Mean prevalence` = mean(Prevalence),
            `Sum prevalence` = sum(Prevalence)) ->
  Prevalence_order_summary

Prevalence_order_summary %>% 
  kable(., digits = c(0, 1, 0)) %>%
  kable_styling(bootstrap_options = c("striped", "hover", "condensed", "responsive"), full_width = F)

Prevalence_phylum_summary %>% 
  filter(`Sum prevalence` < 10) %>% 
  select(Phylum) %>% 
  map(as.character) %>% 
  unlist() ->
  filterPhyla

Ps_obj_filt2 <- subset_taxa(Ps_obj_filt, !Phylum %in% filterPhyla)
Taxonomy.bs %<>% 
  filter(Taxonomy.bs$Name %in% row.names(Ps_obj_filt2@tax_table))
sample_data(Ps_obj_filt2)$Lib.size <- rowSums(otu_table(Ps_obj_filt2))
print(Ps_obj_filt)

print(Ps_obj_filt2)

# Subset to the remaining phyla
prevdf_phylum_filt <- subset(prevdf, Phylum %in% get_taxa_unique(Ps_obj_filt2, "Phylum"))
ggplot(prevdf_phylum_filt,
       aes(TotalAbundance, Prevalence / nsamples(Ps_obj_filt2), color = Phylum)) +
  # Include a guess for parameter
  geom_hline(yintercept = 0.05,
             alpha = 0.5,
             linetype = 2) + geom_point(size = 2, alpha = 0.7) +
  scale_x_log10() +  xlab("Total Abundance") + ylab("Prevalence [Frac. Samples]") +
  facet_wrap( ~ Phylum) + theme(legend.position = "none")

# Subset to the remaining phyla
prevdf_order_filt <- subset(prevdf, Order %in% get_taxa_unique(Ps_obj_filt2, "Order"))

# grab the top 30 most abundant orders
prevdf_order_filt %>% 
  group_by(Order) %>%
  summarise(Combined.abundance = sum(TotalAbundance)) %>% 
  arrange(desc(Combined.abundance)) %>% 
  .[1:30, "Order"]  ->
  Orders2plot

prevdf_order_filt2 <- subset(prevdf, Order %in% Orders2plot$Order)

ggplot(prevdf_order_filt2,
       aes(TotalAbundance, Prevalence / nsamples(Ps_obj_filt2), color = Order)) +
  # Include a guess for parameter
  geom_hline(yintercept = 0.05,
             alpha = 0.5,
             linetype = 2) + geom_point(size = 2, alpha = 0.7) +
  scale_x_log10() +  xlab("Total Abundance") + ylab("Prevalence [Frac. Samples]") +
  facet_wrap( ~ Order) + theme(legend.position = "none")

Ps_obj_filt_Teas <- subset_samples(Ps_obj_filt2, Type != "Soil")
prevdf_teas <- apply(X = otu_table(Ps_obj_filt_Teas),
                 MARGIN = ifelse(taxa_are_rows(Ps_obj_filt_Teas), yes = 1, no = 2),
                 FUN = function(x){sum(x > 0)})
# Add taxonomy and total read counts to this data.frame
prevdf_teas <- data.frame(Prevalence = prevdf_teas,
                      TotalAbundance = taxa_sums(Ps_obj_filt_Teas),
                      tax_table(Ps_obj_filt_Teas))

# Subset to the remaining phyla
prevdf_phylum_filt <- subset(prevdf_teas, Phylum %in% get_taxa_unique(Ps_obj_filt_Teas, "Phylum"))
ggplot(prevdf_phylum_filt,
       aes(TotalAbundance, Prevalence / nsamples(Ps_obj_filt_Teas), color = Phylum)) +
  # Include a guess for parameter
  geom_hline(yintercept = 0.05,
             alpha = 0.5,
             linetype = 2) + geom_point(size = 2, alpha = 0.7) +
  scale_x_log10() +  xlab("Total Abundance") + ylab("Prevalence [Frac. Samples]") +
  facet_wrap( ~ Phylum) + theme(legend.position = "none")

# Subset to the remaining phyla
prevdf_order_filt <- subset(prevdf_teas, Order %in% get_taxa_unique(Ps_obj_filt_Teas, "Order"))

# grab the top 30 most abundant orders
prevdf_order_filt %>% 
  group_by(Order) %>%
  summarise(Combined.abundance = sum(TotalAbundance)) %>% 
  arrange(desc(Combined.abundance)) %>% 
  .[1:30, "Order"]  ->
  Orders2plot

prevdf_order_filt2 <- subset(prevdf, Order %in% Orders2plot$Order)

ggplot(prevdf_order_filt2,
       aes(TotalAbundance, Prevalence / nsamples(Ps_obj_filt_Teas), color = Order)) +
  # Include a guess for parameter
  geom_hline(yintercept = 0.05,
             alpha = 0.5,
             linetype = 2) + geom_point(size = 2, alpha = 0.7) +
  scale_x_log10() +  xlab("Total Abundance") + ylab("Prevalence [Frac. Samples]") +
  facet_wrap( ~ Order) + theme(legend.position = "none")

# Define prevalence threshold as 5% of total samples
prevalenceThreshold <- 0.05 * nsamples(Ps_obj_filt)
prevalenceThreshold

# Execute prevalence filter, using `prune_taxa()` function
keepTaxa <-
  row.names(prevdf_phylum_filt)[(prevdf_phylum_filt$Prevalence >= prevalenceThreshold)]
Ps_obj_filt3 <- prune_taxa(keepTaxa, Ps_obj_filt2)
Taxonomy.bs %<>% 
  filter(Taxonomy.bs$Name %in% row.names(Ps_obj_filt3@tax_table))
sample_data(Ps_obj_filt3)$Lib.size <- rowSums(otu_table(Ps_obj_filt3))
print(Ps_obj_filt2)

print(Ps_obj_filt3)

prevdf_phylum_filt %>% 
  arrange(., Prevalence) %>%  
  group_by(Prevalence > prevalenceThreshold) %>% 
  summarise(Abundance = sum(TotalAbundance)) %>%
  mutate(`Rel. Ab.` = percent(Abundance / sum(Abundance))) %>% 
  kable(., digits = c(0, 1, 0)) %>%
  kable_styling(bootstrap_options = c("striped", "hover", "condensed", "responsive"), full_width = F)

Seq_file <- "DADA2.Seqs.fa"
readDNAStringSet(paste0(data_path, Seq_file)) %>% 
  .[taxa_names(Ps_obj_filt3)] %>%  
  writeXStringSet(., filepath = paste0(data_path, str_remove(Seq_file, ".fa*"), "_filtered.fa"), format = "fasta", width = 1000)

# Subset to the remaining phyla
prevdf_phylum_filt <- subset(prevdf, Phylum %in% get_taxa_unique(Ps_obj_filt3, "Phylum"))
ggplot(prevdf_phylum_filt,
       aes(TotalAbundance, Prevalence / nsamples(Ps_obj_filt3), color = Phylum)) +
  # Include a guess for parameter
  geom_hline(yintercept = 0.05,
             alpha = 0.5,
             linetype = 2) + geom_point(size = 2, alpha = 0.7) +
  scale_x_log10() +  xlab("Total Abundance") + ylab("Prevalence [Frac. Samples]") +
  facet_wrap( ~ Phylum) + theme(legend.position = "none")

# Subset to the remaining phyla
prevdf_order_filt <- subset(prevdf, Order %in% get_taxa_unique(Ps_obj_filt3, "Order"))

# grab the top 30 most abundant orders
prevdf_order_filt %>% 
  group_by(Order) %>%
  summarise(Combined.abundance = sum(TotalAbundance)) %>% 
  arrange(desc(Combined.abundance)) %>% 
  .[1:30, "Order"]  ->
  Orders2plot

prevdf_order_filt2 <- subset(prevdf, Order %in% Orders2plot$Order)

ggplot(prevdf_order_filt2,
       aes(TotalAbundance, Prevalence / nsamples(Ps_obj_filt3), color = Order)) +
  # Include a guess for parameter
  geom_hline(yintercept = 0.05,
             alpha = 0.5,
             linetype = 2) + geom_point(size = 2, alpha = 0.7) +
  scale_x_log10() +  xlab("Total Abundance") + ylab("Prevalence [Frac. Samples]") +
  facet_wrap( ~ Order) + theme(legend.position = "none")

# Subset to the remaining phyla
prevdf_phylum_filt_teas <- subset(prevdf_teas, Phylum %in%  get_taxa_unique(Ps_obj_filt_Teas, "Phylum"))
ggplot(prevdf_phylum_filt,
       aes(TotalAbundance, Prevalence / nsamples(Ps_obj_filt_Teas), color = Phylum)) +
  # Include a guess for parameter
  geom_hline(yintercept = 0.05,
             alpha = 0.5,
             linetype = 2) + geom_point(size = 2, alpha = 0.7) +
  scale_x_log10() +  xlab("Total Abundance") + ylab("Prevalence [Frac. Samples]") +
  facet_wrap( ~ Phylum) + theme(legend.position = "none")

# Subset to the remaining phyla
prevdf_order_filt <- subset(prevdf_teas, Order %in% get_taxa_unique(Ps_obj_filt_Teas, "Order"))

# grab the top 30 most abundant orders
prevdf_order_filt %>% 
  group_by(Order) %>%
  summarise(Combined.abundance = sum(TotalAbundance)) %>% 
  arrange(desc(Combined.abundance)) %>% 
  .[1:30, "Order"]  ->
  Orders2plot

prevdf_order_filt2 <- subset(prevdf, Order %in% Orders2plot$Order)

ggplot(prevdf_order_filt2,
       aes(TotalAbundance, Prevalence / nsamples(Ps_obj_filt_Teas), color = Order)) +
  # Include a guess for parameter
  geom_hline(yintercept = 0.05,
             alpha = 0.5,
             linetype = 2) + geom_point(size = 2, alpha = 0.7) +
  scale_x_log10() +  xlab("Total Abundance") + ylab("Prevalence [Frac. Samples]") +
  facet_wrap( ~ Order) + theme(legend.position = "none")

# remove samples with reads < min_lib_size
Ps_obj_filt3 %>%
  subset_samples(., sample_sums(Ps_obj_filt3) > min_lib_size) %>%
  filter_taxa(., function(x)
    sum(x) > 0, TRUE) ->
  # transform_sample_counts(., function(x)
    # x / sum(x) * 100) ->
  Ps_obj_filt3_subset

Ps_obj_filt3_subset %>% 
  get_variable() %>% 
  mutate_at(., "Sample.type", 
            ~fct_relevel(., levels = c("Soil", "Green tea", "Rooibos"))) %>% 
    mutate_at(., "Season", 
            ~fct_relevel(., levels = c("Winter", "Spring", "Summer", "Autumn"))) %>% 
  arrange(Field, Sample.type, Season, Replicate) %>% 
  pull(Description) %>% 
  as.character() ->
  Sample.order

Ps_obj_filt3_subset_ra <- transform_sample_counts(Ps_obj_filt3_subset, function(x){x / sum(x)} * 100)

# grabthe top 100 most abundant OTUs
Ps_obj_filt3_subset_100 <-
  prune_taxa(names(sort(taxa_sums(Ps_obj_filt3_subset_ra), TRUE)[1:100]), Ps_obj_filt3_subset_ra)
plot_heatmap(
  Ps_obj_filt3_subset_100,
  method = NULL,
  distance = NULL,
  sample.label = "Description",
  taxa.label = "Order",
  sample.order = Sample.order,
  low = "#000033",
  high = "#FF3300"
)

Ps_obj_filt3_subset_ra %>% 
  subset_taxa(., Phylum == "Ascomycota") %>% 
  transform_sample_counts(., function(x) x / sum(x) * 100) ->
  Ps_obj_filt3_subset_asco
plot_heatmap(
  Ps_obj_filt3_subset_asco,
  method = NULL,
  distance = NULL,
  sample.label = "Description",
  sample.order = Sample.order,
  taxa.label = "Order",
  low = "#000033",
  high = "#FF3300"
)

Ps_obj_filt3_subset_ra %>% 
  subset_taxa(., Phylum == "Basidiomycota") %>% 
  transform_sample_counts(., function(x) x / sum(x) * 100) ->
  Ps_obj_filt3_subset_basido
plot_heatmap(
  Ps_obj_filt3_subset_basido,
  method = NULL,
  distance = NULL,
  sample.label = "Description",
  sample.order = Sample.order,
  taxa.label = "Order",
  low = "#000033",
  high = "#FF3300"
)

Ps_obj_filt3_subset_ra %>% 
  subset_taxa(., Phylum == "Chytridiomycota") %>% 
  transform_sample_counts(., function(x) x / sum(x) * 100) ->
  Ps_obj_filt3_subset_chytri
plot_heatmap(
  Ps_obj_filt3_subset_chytri,
  method = NULL,
  distance = NULL,
  sample.label = "Description",
  sample.order = Sample.order,
  taxa.label = "Order",
  low = "#000033",
  high = "#FF3300"
) 

Ps_obj_filt3_subset_ra %>% 
  subset_taxa(., Phylum == "Mortierellomycota") %>% 
  transform_sample_counts(., function(x) x / sum(x) * 100) ->
  Ps_obj_filt3_subset_morti

plot_heatmap(
  Ps_obj_filt3_subset_morti,
  method = NULL,
  distance = NULL,
  sample.label = "Description",
  sample.order = Sample.order,
  taxa.label = "Phylum",
  low = "#000033",
  high = "#FF3300"
)

Ps_obj_filt3_subset_ra %>% 
  subset_taxa(., Order == "Helotiales") %>% 
  transform_sample_counts(., function(x) x / sum(x) * 100) ->
  Ps_obj_filt3_subset_helo
plot_heatmap(
  Ps_obj_filt3_subset_helo,
  method = NULL,
  distance = NULL,
  sample.label = "Description",
  sample.order = Sample.order,
  taxa.label = "Order",
  low = "#000033",
  high = "#FF3300"
)

Ps_obj_filt3_subset_ra %>% 
  subset_taxa(., Order == "Hypocreales") %>% 
  transform_sample_counts(., function(x) x / sum(x) * 100) ->
  Ps_obj_filt3_subset_hypo
plot_heatmap(
  Ps_obj_filt3_subset_hypo,
  method = NULL,
  distance = NULL,
  sample.label = "Description",
  sample.order = Sample.order,
  taxa.label = "Order",
  low = "#000033",
  high = "#FF3300"
)

Ps_obj_filt3_subset_ra %>% 
  subset_taxa(., Order == "Pleosporales") %>% 
  transform_sample_counts(., function(x) x / sum(x) * 100) ->
  Ps_obj_filt3_subset_pleo
plot_heatmap(
  Ps_obj_filt3_subset_pleo,
  method = NULL,
  distance = NULL,
  sample.label = "Description",
  sample.order = Sample.order,
  taxa.label = "Order",
  low = "#000033",
  high = "#FF3300"
)

Ps_obj_filt3_subset_ra %>% 
  tax_glom(., "Order", NArm = TRUE) ->
  Ps_obj_filt_glom

plot_heatmap(
  Ps_obj_filt_glom,
  # method = "NMDS",
  # distance = "bray",
  sample.order = Sample.order,
  sample.label = "Description",
  taxa.label = "Order",
  taxa.order = "Order",
  low = "#000033",
  high = "#FF3300"
) + theme_bw(base_size = 20) + theme(axis.text.x = element_text(hjust = 1.0, angle = 90.0))

PlotAbundance(Ps_obj_filt3_subset_ra, phylum2plot = "Ascomycota")

# plotBefore <- PlotAbundance(Ps_obj_filt3_subset, taxa = "Proteobacteria")
# plotAfter <- PlotAbundance(Ps_obj_filt3_subset_ra, taxa = "Proteobacteria")
# Combine each plot into one graphic.
# grid.arrange(nrow = 2, plotBefore, plotAfter)

Ps_obj_filt3_subset_ra_taxa <- subset_taxa(Ps_obj_filt3_subset_ra, Order == "Helotiales")
PlotAbundance(Ps_obj_filt3_subset_ra_taxa, phylum2plot = "Ascomycota", Facet = "Genus")

Ps_obj_filt3_subset_df <- psmelt(Ps_obj_filt3_subset)

# Ps_obj_filt3_subset_df %>% 
#   filter(Species == "Epibolus pulchripes") ->
#   Ps_obj_filt3_subset_df_EP 
# PlotViolin(Ps_obj_filt3_subset_df_EP)

PlotViolin(Ps_obj_filt3_subset_df)

Ps_obj_filt3_glom <- tax_glom(Ps_obj_filt3_subset_ra, 
                             "Order", 
                             NArm = TRUE)
Ps_obj_filt3_glom_DF <- psmelt(Ps_obj_filt3_glom)
Ps_obj_filt3_glom_DF$Order %<>% as.character()

# group dataframe by Phylum, calculate median rel. abundance
Ps_obj_filt3_glom_DF %>%
  group_by(Order) %>%
  summarise(median = median(Abundance)) ->
  medians

# find Phyla whose rel. abund. is less than 0.5%
Rare_phyla <- medians[medians$median <= 0.005, ]$Order

# change their name to "Rare"
Ps_obj_filt3_glom_DF[Ps_obj_filt3_glom_DF$Order %in% Rare_phyla, ]$Order <- 'Rare'
# re-group
Ps_obj_filt3_glom_DF %>%
  group_by(Sample, Sample.name, Order, Sample.type, Field) %>%
  summarise(Abundance = sum(Abundance)) ->
  Ps_obj_filt3_glom_DF_2plot

# ab.taxonomy$Freq <- sqrt(ab.taxonomy$Freq)
# Ps_obj_filt3_glom_rel_DF$Phylum %<>% sub("unclassified", "Unclassified", .)
# Ps_obj_filt3_glom_rel_DF$Phylum %<>% sub("uncultured", "Unclassified", .)

Ps_obj_filt3_glom_DF_2plot %>% 
  group_by(Sample) %>% 
  filter(Order == "Rare") %>% 
  summarise(`Rares (%)` = sum(Abundance * 100)) -> 
  Rares
# Percentage of reads classified as rare 
Rares %>%
  kable(., digits = c(2), caption = "Percentage of reads per sample classified as rare:") %>%
  kable_styling(bootstrap_options = c("striped", "hover", "condensed", "responsive"), full_width = F)

sample_order <- match(Rares$Sample, row.names(get_variable(Ps_obj_filt3_glom)))
Rares %<>% arrange(., sample_order)

Rares %>% 
  cbind(., get_variable(Ps_obj_filt3_glom)) %>% 
  group_by(Sample.type) %>% 
  summarise(`Rares (%)` = mean(`Rares (%)`)) -> 
  Rares_merged

# Percentage of reads classified as rare 
Rares_merged %>%
  kable(., digits = c(2), caption = "Percentage of reads per sample classified as rare:") %>%
  kable_styling(bootstrap_options = c("striped", "hover", "condensed", "responsive"), full_width = F)

Ps_obj_filt3_glom_DF_2plot %>% 
  group_by(Order) %>% 
  summarise(sum.Taxa = sum(Abundance)) %>% 
  arrange(desc(sum.Taxa)) -> Taxa.rank
Ps_obj_filt3_glom_DF_2plot$Order %<>% 
  factor(., levels = Taxa.rank$Order) %>% 
  fct_relevel(., "Rare", after = Inf)
  
p_taxa_box <-
  ggplot(Ps_obj_filt3_glom_DF_2plot, aes(x = Order, y = (Abundance))) +
  geom_boxplot(aes(group = interaction(Order, Sample.type)), position = position_dodge(width = 0.9), fatten = 1) +
  geom_point(
    aes(colour = Sample.type),
    position = position_jitterdodge(dodge.width = 1),
    alpha = 1 / 2,
    stroke = 0,
    size = 2
  ) +
  # scale_colour_manual(values = pom4, name = "") +
  theme_cowplot(font_size = 11, font_family = f_name) +
  labs(x = NULL, y = "Relative abundance (%)") +
  guides(colour = guide_legend(override.aes = list(size = 5))) +
  facet_grid(Field ~ .) +
  background_grid(major = "xy",
                  minor = "none") +
  theme(axis.text.x = element_text(
    angle = 45,
    vjust = 0.9,
    hjust = 0.9
  ))
print(p_taxa_box)

dir.create(paste0(data_path, "Krona/"))
for (i in seq(nsamples(Ps_obj_filt3_subset))) {
  sample_data <-
    data.frame(Abundance = get_taxa(Ps_obj_filt3_subset, i), as(tax_table(Ps_obj_filt3_subset), "matrix"))
  sample_data <- sample_data[sample_data[, 1] > 0, ]
  write_tsv(sample_data, paste0(data_path, "Krona/", sample_names(Ps_obj_filt3_subset)[i], ".tsv"))
}

sample_data(Ps_obj_filt3_subset)$Sample.type %<>% fct_recode(., Green_tea = "Green tea")

sample_data(Ps_obj_filt3_subset)$Sample.type_field_season <- paste0(
  get_variable(Ps_obj_filt3_subset, "Sample.type"),
  "_",
  get_variable(Ps_obj_filt3_subset, "Field"),
  "_",
  get_variable(Ps_obj_filt3_subset, "Season")
)

Ps_obj_filt3_subset %>%
  phyloseq::merge_samples(., "Sample.type_field_season", fun = mean) ->
  merged.Ps_obj

for (i in seq(nsamples(merged.Ps_obj))) {
  sample_data <-
    data.frame(Abundance = get_taxa(merged.Ps_obj, i), as(tax_table(merged.Ps_obj), "matrix"))
  sample_data <- sample_data[sample_data[, 1] > 0, ]
  write_tsv(sample_data, paste0(data_path, "Krona/", sample_names(merged.Ps_obj)[i], ".tsv"))
}

list.files(paste0(data_path, "Krona/"), full.names = TRUE) %>%
  paste(., collapse = " ") %>%
  paste0("/usr/local/bin/ktImportText ", .,
         " -o ",
         data_path,
         "Krona/TeaTime_Krona.html") %>%
  system()

saveRDS(Ps_obj_filt, file = paste0(data_path, Proj_name, "ITS_filt.RDS"))
saveRDS(Ps_obj_filt3, file = paste0(data_path, Proj_name, "ITS_filt3.RDS"))

sessioninfo::session_info() %>%
  details::details(
    summary = 'Current session info',
    open    = TRUE
 )